For many years scientists yearned for the possibility of performing flow cytometry to analyse small bio-nanoparticles that are too small to be measured by conventional and high sensitivity instruments. These entities, extracellular vesicles, gene therapy vectors, viruses and drug delivery particles, are promised to become the next generation of therapeutics, but they have been hard to handle and characterise due to their small size and biological or chemical heterogeneity. There is therefore a strong case for bringing flow cytometry capability to the sub-200nm scale. NanoFCM has developed the NanoAnalyzer platform that now enables true flow-cytometry measurement at the sub-micron scale, and down to particle sizes unreachable by any other flow cytometers (10-40nm depending on the nature of the substrate). Nano-flow cytometry, the technology that underpins the NanoAnalyzer, removes bias and uncertainty stemming from the use of fluorescence signal triggering by using its highly sensitive side-scatter channel to trigger particle events. The single-particle nature of the measurement prevents uncontrolled swarming events, reinforcing data integrity. High resolution of both scatter and fluorescence channels allows the assessment of subpopulations, based on size or on bio-chemical properties. Nano-flow cytometry’s ability to measure simultaneously a (bio)-nanoparticle population for size, size distribution and bio-chemical properties on a single instrument dramatically improves data quality and throughput compared to the traditional, multiple-techniques approach involving particle characterisation and counting (DLS, NTA, RPS), combined with chemical and biological function assessment (ELISA, Western Blot, Flow Cytometry, PCR). Quantitative measurement of the active and contaminant particles in a single preparation opens up the possibility of characterisation-based nanomedicine regulatory approval, and allows the conduct of large-scale clinical studies. From the research laboratory to the quality control department, NanoFCM delivers comprehensive bio-nano analysis.

1. Anindita Guha, M.Sc Cellular and Molecular Oncology
Product Specialist, SG Instruments Pvt. Ltd., New Delhi
anindita@sg-instruments.com

2. Implementing strategies for single-molecule fluorescence detection in a sheathed flow,
NanoFCM provides a versatile and powerful platform —— Flow NanoAnalyzer for the
multiparameter analysis of functional nanoparticles (7-1000 nm) at the single-particle level.

3. Label-Free Analysis of Single Extracellular Vesicles for the Size Distribution and
Concentration Determination
Much attention has been focused on the molecular characterization of EV content, primarily mRNAs, miRNAs and proteins, and on the specific
markers exposed on the lipid bilayers that determine specific interactions with target cells. However, it has been suggested that physical properties
of the particles may also affect the behavior of EVs, such as the way they mediate intercellular communication. Employing monodisperse silica
nanospheres as the size standards, the side scatter intensity of individual EVs can be converted to the size information. In this data,
approximately 65% of the EVs fall in the size range of 30-100 nm and 86% in the size range of 30-200 nm.

4. Extracellular Vesicles for the Early Diagnosis of Cancer
EVs are associated to many pathological
conditions such as thrombosis, haemostasis,
inflammation, sickle cell disease and malaria, they
may serve as biomarkers of diseases and
therapeutic targets. A deeper understanding of the
cargo molecules present in EVs obtained from
patients with cancers, will aid in the identification
of novel diagnostic and prognostic biomarkers,
and can potentially lead to the discovery of new
therapeutic targets. The detection of biomarkers
in body fluids has major advantages over the use
of tissue markers, which most often require
invasive biopsies that can be difficult to perform
and potentially dangerous. Employing CD147 as
biomarker, the NanoFCM is able to
discriminate the EVs isolated from cancer cell
lines and from normal colon cell lines. The
results agree well with Western blotting methods,
suggesting its potential utility in cancer diagnosis.
It enables discrimination of EVs extracted from
plasma of colorectal cancer patients and healthy
donors after immunofluorescent labeling.

5. Tracking in vitro EV behavior for Autoimmune and Neurodegenerative disorders
EVs have physiological and potential pathological effects in autoimmunity and neurodegenerative diseases: the secretion and release of EVs are
strictly regulated processes, and the secretion and release processes of EVs are different under physiological and pathological conditions.
Electrical stimulation as a clean and effective method is an ideal way to regulate the secretion of therapeutic EVs compared to chemical
stimulation that lacks specificity and has side effects. In this study, the most abundant and heterogeneous astrocytes in the central nervous
system (CNS) were selected, to which electrical stimulation with different parameters were applied while the distribution of EVs in
whose supernatants were detected by aquaporin AQP4. Flow NanoAnalyzer were used to characterize the particle size distribution and
surface protein of EVs under different stimulation conditions, thus further revealing the relationship between electrical stimulation and secretion
pathways of EVs. Under the condition of no external electrical stimulation, AQP4-positive EVs were clearly divided into two groups,
corresponding to the particle size range of exosomes and microvesicles.

6. Nucleic Acids and Lipid Analysis of Extracellular Vesicles from Plasma
Vesicular cargoes, including RNA, DNA, proteins,
lipids, and metabolites, seem to be found inside
and on the surface of EVs. This EV cargo can be
transferred to recipient cells, resulting in a
pleiotropic response. Insights into the function
of EVs can be obtained either by measuring the
composition or by assays in which the function
can be evaluated.
Here, SYTO RNASelect Green Fluorescent Stain
is used to label the mRNA and miRNA of the
EVs, PKH26 is employed to label the lipid
structure of the EVs.

7. Autofluorescence Quantification of Single Bacteria
Flow NanoAnalyzer can explicitly detect the autofluorescence of a single bacterium- the green autofluorescence mainly originates from
oxidized form of endogenous flavin. Among the eight bacterial strains tested, it is found that the signal of bacterial autofluorescence is
closely related with bacterial size. Bacterial autofluorescence is quantified in units of FITC equivalents by using fluorescent nanoparticles
with known FITC equivalents as the quantitative calibration standards.

8. Label-Free Detection of Single Viruses
The virus used in this experiment is levivirus MS2- non-enveloped, spherical virion about 27 nm in diameter, and the genome is
monopartite, linear ssRNA (+) about 3.5 kb in size. The signal-to-noise (S/N) ratio calculated as the average burst height of all the
nanoparticles detected in 1 min divided by the standard deviation of the background signal (noise), is 11 for the MS2 viruses, indicating
that the Flow NanoAnalyzer provides exceptional sensitivity in discerning MS2 viruses against the background noise.

9. Rapid Detection of Resistant Bacteria Based on β-lactamase Activity
Among many molecular mechanisms that confer antibiotic resistance, production of β-lactamase that catalyze the hydrolysis of β-lactam
antibiotics is a major and threatening mechanism. It has been reported that individuals could be simultaneously infected with multiple
strains of different susceptibility levels. Traditional detection method cannot detect minority population of antibiotic-resistant bacteria.
NanoFCM allows rapid single-cell detection and quantitative observation of the resistant bacterial population (down to 5%)
through fluorescent probe LBRL1.

10. Clinical Diagnosis of Bacterial Infection and Resistance
Individuals could be simultaneously infected with multiple strains of different susceptibility levels, and the population of resistant
bacteria could be very low. However, if the minority population of resistant bacteria cannot be detected in time, an inappropriate
prescription of antibiotics is usually a result. Through fluorescent immunolabeling and nucleic acid staining, detection of minority
population of antibiotic-resistant bacteria is achieved. This method allows real-time track of the dynamic population change of
antibiotic-resistant bacteria with and without antibiotics. Detection of antibiotic-resistant infection in clinical urine samples is
achieved without cultivation, and the bacterial load of susceptible and resistant strains is quantified.

11. Absolute and Simultaneous Quantification of Specific Pathogenic
Strain and Total Bacterial Cells
By integrating antigen and nucleic acid double fluorescence staining, a sensitive approach for the rapid, absolute, and simultaneous
quantification of specific pathogenic strain and total bacteria cells in mixture is developed. Here Alexa Fluor 647-R-PE is used as the
fluorescent probe for the monoclonal antibody of pathogenic E. coli O157:H7, the green fluorophore SYTO 9 is used to stain all the
bacterial cells. Double-stained E. coli O157:H7, can be specifically identified and enumerated using two-color fluorescence
coincidence detection, while non-pathogenic bacteria can be quantified by green fluorescence detection.

12. Identification of Mitochondria-Targeting Anticancer Drugs
Mitochondria play a pivotal role in determining the “point-of-no-return” of the apoptotic process. Therefore, anticancer drugs that directly target
mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. By labeling with
DiOC6(3), Flow NanoAnalyzer can monitor the change of mitochondrial membrane potential. This method serves an in vitro strategy to quickly
identify the therapeutic agents that induce apoptosis via directly affecting mitochondria, and side scatter detection can reveal the change of internal
contents upon drug treatment at the single-organelle level with high resolution.

13. Multiparameter Quantification of Liposomes at Single Particle Level
A liposome is a tiny vesicle, made out of the similar material as a
cell membrane. Drug-encapsulated liposomes have been
considered the most clinically acceptable drug-delivery systems.
Here, Flow NanoAnalyzer was used to simultaneously detect
the side-scatter and fluorescence signals generated by
individual liposome particles at a speed up to 10,000
nanoparticles/min. To cope with the size dependence of the
refractive index of liposomes, different sizes of doxorubicin-
loaded liposomes were fabricated and characterized to serve as
the calibration standards for the measurement of both particle
size and drug content. This method provides a highly practical
platform for the characterization of liposome vesicles.

14. Discrimination and Size Measurement of Viruses in a Mixture

15. Extracellular Vesicles Encapsulated Oncolytic Viruses for the
Treatment of Tumor Diseases
Extracellular vesicles have the ability to pass the blood-brain barrier, and can also target tumor cells through specific markers,
which in turn becomes one of the most ideal carriers for drug treatment of tumor diseases. Oncolytic viruses (OA) are a type of
tumor-killing virus with replication ability, which selectively infect tumor cells by deactivating tumor suppressor genes in target cells,
replicating themselves in their cytoplasm and eventually destroying them. It also stimulates the immune response, attracting more
immune cells to continue killing residual cancer cells. The exosome-encapsulated oncolytic adenovirus can resist the innate and
acquired immunity of the human body, and can specifically infect tumor cells and self-replicate to form a large number of viruses
that specifically infect tumors, thereby killing tumor cells. Based on nucleic acid staining, Flow NanoAnalyzer allows the
determination of the encapsulation efficiency of oncolytic adenoviruses, which was 59.9% in this case.

16. Analysis of
Lentiviral Particle
Concentration and
Nucleic Acid
Encapsulation
Efficiency
Chimeric antigen receptor (CAR) T cell therapy for B cell malignancies have fueled an increasing number of clinical trials and the US FDA’s
first approval of cell therapies for cancer treatment. Lentivirus is the most commonly used viral vector in CAR-T cell therapy. A complete
CAR-T cell process includes the preparation of lentiviral vectors and the production of CAR-T cell products. Accurate measurement of
lentivirus concentrations will have great impact on the process optimization, and further improve quality assurance. Plaque titer and
TCID50 method are the most classical approaches for concentration measurement of viruses, however, they quantify only those which
cause visible cell-damage thus exclude the viruses without infectivity. With NanoFCM, the concentration of intact lentiviral particles
was 3.06 × 10^11 particles/mL, and the nucleic acid encapsulation efficiency of lentivirus was 61.7%.

17. High-Throughput Single-Cell Analysis of Low Copy Number Protein
β-galactosidase (β-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Built upon the
sensitivity and speed of the instrument and the good cell retention of the hydrolysis products of C12FDG, β-gal is detected at single
bacterial cell level. Combining with the quantitative MUG fluorometric assay and the rapid bacterial enumeration on the instrument, the
distribution profile of β-gal expression is quantified in protein copy numbers per cell. In addition to the β-gal gene reporter, fluorescent
proteins or tetracysteine tags that can be genetically fused with the target protein would further expand the scope of the instrument in the
investigation of low abundance gene expression and regulation.

18. Size Differentiation and Absolute Quantification of Gold Nanoparticles
Gold nanoparticles (GNPs) have recently attracted enormous attention in medical, bioanalytical, and catalytic applications due to their unique optical,
physical, and chemical properties. Accurate size and concentration measurements of GNPs are vital to the quality control of GNP synthesis, surface
functionalization, and assay development. In contrast to the numerous methods for GNP size measurement, there is no efficient method for the
accurate quantification of GNPs. The concentration of GNPs is usually calculated according to the GNP size measured by TEM and the gold content
analyzed by inductive coupled plasma mass spectrometry (ICP-MS). In addition to requiring expensive equipment, sample preparation is time-
consuming. A standard-free absolute quantitative analysis method for particle concentration was developed on the Flow NanoAnalyzer through the
measurement of single particle count and sample volume flow per unit time.

19. Rapid and Quantitative Measurement of Single Quantum Dots
Semiconducting quantum dots (QDs) are finding a wide range of biomedical applications (bioimaging, nano-drug-carriers, theranostics)
due to their intense fluorescence brightness and long-term photostability. Here, precise quantification of the fluorescence intensity of single
QDs on the Flow NanoAnalyzer is reported. By analysis of thousands of QDs individually in 1 min, intrinsic polydispersity was quickly
revealed in a statistically robust manner.

20. The Role of Extracellular Vesicles in Immune Response
Endosomal Toll-like receptors (TLRs) mediate intracellular innate immunity by recognizing DNA and RNA sequences. Recent studies have reported
the role of extracellular vesicles (EVs) that transfer various nucleic acids in the uptake of TLR activating molecules, triggering speculation about the
possible role of EVs in innate immune surveillance. This study demonstrates that when macrophages are stimulated with the TLR9 agonist
CpG oligodeoxynucleotide (ODN), secreted EVs transport ODN into the original macrophages and induce the release of the chemokine
TNF-α. In addition, these EVs transfer Cdc42 into recipient cells, thereby further enhancing their cellular uptake. ODN and Cdc42 play a
synergistic role in the transmission of TLR9-activated macrophages via EVs to primordial cells in the propagation of intracellular immune responses,
suggesting a general mechanism of EVs mediated pathogen-associated molecular pattern uptake.

21. Identify Glypican-1 Associated with Exosomes from Pancreatic Cancer Cells and Serum from
Patients with Pancreatic Cancer
In the early work, the research team found that the number of Glypican-1 (GPC1)-positive exosomes in the circulatory system of patients with
pancreatic cancer was significantly higher than that of normal people, and in follow-up studies they found that in addition to pancreatic cancer, breast
cancer, GPC1-positive exosomes are also enriched in colorectal cancer, and esophageal cancer, indicating that GPC1 is expected to become a
marker for early diagnosis of a variety of cancers. In this study, immunofluorescence staining was performed on exosomes in patients with
pancreatic cancer, pancreatic benign disease, and normal human serum. The multi-parameter analysis of the Flow NanoAnalyzer was used
to verify the overexpression of exosomal GPC1 from pancreatic cancer patients at the level of individual exosomes for the first time.

22. Characterization of Doxorubicin-Carrying Doxoves
Doxil (doxorubicin-carrying liposomes) is the first FDA-
approved nanomedicine (1995), and DLS and cryo-
TEM are the two most commonly used methods for size
analysis. While DLS is not appropriate for
heterogeneous samples, the 3D reconstruction of cryo-
TEM usually takes 2-3 days. Doxoves, a research-
grade product of PEGylated liposomal doxorubicin
whose physical characteristics and pharmacokinetics
are comparable to those of Doxil, is analyzed as a
model system. Monodisperse silica nanoparticles
are used as the standard to calibrate the size
measurement of the Doxoves nanoparticles based
on their SS burst areas. A substantial amount of
variation in both size and doxorubicin content can
be observed among individual particles.

23. Single-Particle Characterization of Theranostic Liposomes with Stimulus Sensing and
Controlled Drug Release Properties
Here, fabricating a reactive oxygen species (ROS)-responsive liposome (Lipo@BODIPY11) and taking it as an example, a strategy for theranostic
nanoparticle characterization by the Flow NanoAnalyzer was developed. Coincident detection of light scatter and fluorescence intensity provided a
measurement for liposome quality assessment. Theranostic performance referred to stimuli-responsive capability and drug release behavior upon ROS
treatment can be obtained in minutes. For ratiometric fluorescence measurement, the Flow NanoAnalyzer is equipped with 488 nm laser, and the
detection channels are side scatter, green fluorescence (FITC), and orange fluorescence (PE), while for ROS sensing and drug release
assessment, a second laser of 642 nm is added, and the detection channels are side scatter, green fluorescence (FITC), and red fluorescence
(710/40 nm).